G protein subunits play a central role in G-protein coupled receptor (GPCR)-mediated signal transduction processes. They act as cofactors in the activation of 7TM receptors and play direct roles in signaling to intracellular targets. GPCR signaling is extremely significant from a therapeutic standpoint;indeed, the majority of marketed pharmaceuticals target the receptor components of GPCRs. Less developed to date is the selective manipulation of intracellular subunit signaling, which could allow for the simultaneous modulation of multiple signaling pathways downstream of 7TM receptors. The commercialization of targeted modulators of subunit signaling would therefore have a tremendous impact on drug discovery paradigms. Professors Alan Smrcka, Burns Blaxall and Jean Bidlack at the University of Rochester Medical Center (URMC) have developed a novel strategy for the selective manipulation of G-protein subunit signaling that blocks - subunit binding to functional protein partners using small molecular weight compounds. They have demonstrated that these compounds are efficacious in animal models of heart failure, inflammation, and morphine-dependent analgesia. Califia Bio, Inc. is collaborating with the URMC team to develop these initial hit molecules into advanced chemical leads. These leads will ultimately be progressed into clinical studies for the treatment of cardiovascular disease and chronic pain. The current URMC hit molecules were identified from the NCI compound collection and lack drug-like characteristics as well as definitive intellectual property protection, which hampers their commercial development. In order to further develop novel small molecule inhibitors of subunit protein interactions, Califia Bio will utilize: 1) SAR gathered by URMC in high- throughput screening campaigns, 2) an X-ray crystal structure of previously identified small helical peptide inhibitor that binds to the subunit hot spot, 3) an X-ray crystal structure of a weak affinity small molecule ligand that binds to the same hot spot, and 4) specifically targeted reversible inhibitors designed to block a key active cysteine in the hot spot. After their initial design, potential inhibitors will be docked into the existing crystal structures and further triaged before synthesis by assessing their drug-like character and potential ADMET issues with proprietary software from Optibrium Ltd. A primary ELISA binding assay will be used to prioritize compounds, followed by secondary assays for inhibition of PLC activation and GRK2 G binding inhibition. -adrenergic receptor (BAR) dysfunction represents a hallmark abnormality of heart failure and interference of G-protein mediated GRK2 activity has been demonstrated to reverse the effects of (BAR) overstimulation. A patentable lead series will be identified, and the potency and selected ADME properties of this series will be optimized utilizing the above mentioned in vitro assays. In phase two, efficacy in animal models will be established and ADME properties will be optimized to allow for advanced preclinical development of potential therapeutics for treatment of heart failure and chronic pain. PUBLIC HEALTH RELEVANCE: This Phase I SBIR proposal is aimed at the discovery of potential small molecule therapeutics to treat heart failure. The mechanism involved may also be of interest for enhancing the efficacy and lessening the side effects of opioids used to treat chronic pain. Heart failure is estimated to be a $2 billion dollar drug market where there is great unmet therapeutic need, with approximately 5.7 million patients in the US, and 670,000 new cases per year. In end stage heart failure, only one half of patients will survive one year, and the only successful treatment is heart transplant, where only approximately 2000 patients per year are lucky enough to receive new hearts.